Phytochemicals for the treatment of mastalgia, endometriosis and HPV-related conditions including cervical dysplasia

ABSTRACT

New compositions are disclosed that comprise the phytochemical Diindolylmethane (DIM), as well as its precursor, Indole-3-carbinol (I3C), and cogener, 2-(Indol-3-ylmethyl)-3,3′diindolylmethane (LTr-1), acceptable carriers and/or excipients. These compositions are administered to prevent or reduce symptoms associated with mastalgia, endometriosis and HPV-related conditions including cervical dysplasia.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of prior applicationSer. No. 09/404,111, filed Sep. 23, 1999, the entire contents of whichare hereby expressly incorporated by reference.

1. FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods forprevention or reduction of symptoms associated with mastalgia,endometriosis and Human Papilloma Virus-related conditions including,but not limited to, cervical dysplasia, by administering indolephytochemicals. Among the indole phytochemicals useful in thecompositions and methods of the invention are diindolylmethane (DIM),and its cogener, 2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTr-1).

2. BACKGROUND OF THE INVENTION

[0003] 2.1. Introduction

[0004] In the typical life-cycle of modern women, the mid-thirties untilthe cessation of menstrual periods is defined as “peri-menopause”. Womenare classified as perimenopausal if menses have been experienced in thelast 12 months, but with irregularity or changes in menstrual flow.During this stage of life women suffer increasing incidence of bothrecurrent breast pain, or “mastalgia”, and endometriosis, describing thepainful condition of persistence of abnormal endometrial tissue in theabdominal cavity. These two conditions, common to the perimenopause, arepoorly understood and presently lack medical therapy that is botheffective and reasonably free of side effects (Prior J C, 1998,“Perimenopause: the complex endocrinology of the menopausal transition”,Endocr. Rev. 19:397-428).

[0005] While a contributing role of estrogen status has been suspectedin these conditions, few consistent abnormalities in endocrine statushave been identified. Circulating estrogen levels are normal in bothmastalgia and endometriosis patients. There is accumulating evidencethat describes diminished progesterone production during theperimenopause that may create a relative “dominance” in the activity ofestrogen.

[0006] However, no one theory or endocrine imbalance explains theoccurrence of mastalgia and endometriosis (Santoro N et al., 1996,“Characteristics of reproductive hormonal dynamics in theperimenopause”, J. Clinical Endocrinology and Metab. 81:1495-501).Similarly, changes in estrogen to progesterone balance in women have notbeen associated with the occurrence of Human Papilloma Virus(HPV)-related cervical dysplasia.

[0007] 2.2. Mastalgia

[0008] Recurrent, cyclical breast pain or “mastalgia” is one of the mostcommon reasons for women's visits to their doctors. It has beenestimated that 50-70% of all women experience significant mastalgia atsome point in their life. In its most common form, mastalgia is achronic condition of recurring pain, which is worse during the few daysbefore menses (Blue J et al., 1998, “Mastalgia review: St. Marks BreastCentre”, New Zealand Medical Journal 111(1059):33-34). Traditionally,treatment choices for mastalgia have ranged from dietary manipulation(caffeine, fat, and alcohol reduction) or evening primrose oil tohormonal medications (bromocriptine and danazol) for severe breast pain.Bromocriptine (Parlodel) and danazol have a response rate of 70%, buthave reported adverse side effects of up to 30-35% (Gateley C A andMansen R E, 1991, “Management of the painful and nodular breast”,British Medical Bulletin 47:284-94; Nazli K et al., 1989, “Controlledtrial of the prolactin inhibitor bromocriptine (Parlodel) in thetreatment of severe cyclical mastalgia”, British Journal of ClinicalPractice 43(9):322-7; Kontosolis K et al., 1997, “Comparison oftamoxifen with danazol for treatment of cyclical mastalgia”, Gynecol.Endocrinol. 11:393-397). The use of medroxyprogesterone acetate tosupport levels of progesterone, possibly low in this condition, providedrelief no better than placebo in a controlled trial (Maddox P R et al.,1990, “A randomized controlled trial of medroxyprogesterone acetate inmastalgia”, Annals of the Royal College of Surgeons of England72(2):71-6).

[0009] The approach of dietary supplementation for mastalgia has beenexplored by earlier investigators. This included the addition of highdoses of evening primrose oil, beta carotene, and vitamin A to the dietof affected women. Evening primrose oil is used by British physicians asan initial intervention to control mastalgia because of its non-hormonalcomposition. Though it has been found to normalize the ratio ofessential fatty acids to saturated fatty acids in the serum of womenwith mastalgia, the therapy requires 3 to 4 months for benefit.Improvements were seen in up to 40% of patients but side effectsincluded bloating and nausea (Maddox P R, 1989, “The management ofmastalgia in the UK”, Hormone Research 32:21-27). Italian researchersexplored the addition of combinations of beta carotene and Vitamin A(retinol) in the management of mastalgia. (Santamaria L et al., 1989,“Beta-carotene supplementation associated with intermittent retinoladministration in the treatment of pre-menopausal mastodynia”, Boll ChimFar 128:284-287). Some success was reported, but the high doses ofretinol required (150-300,000 I.U per day) are in the range associatedwith significant side effects which include headache, skin lip and mouthdryness, nausea, dizziness, and alopecia. Based on the common occurrenceof mastalgia as a disorder in women, the need exists for more effectivetherapy with acceptable risks and side effects (Ashley B, 1998,“Mastalgia”, Lippincotts Primary Care Practice 2(2):189-93).

[0010] 2.3. Endometriosis

[0011] Endometriosis is a disease that affects as many as 15% of fertilewomen and up to 50% of infertile women. Its occurrence increases withage and is greatest in the perimenopausal years (Tzingounis V A andCardamakis E, 1997, “Modem approach to endometriosis”, Annals of the NewYork Academy of Sciences 816:320-330). Endometriosis refers to thepresence of functional endometrial glands and stroma in abnormallocations outside the uterine cavity. Despite extensive research, thenatural history and pathogenesis of endometriosis is still poorlyunderstood and remains controversial. As with mastalgia, mosttherapeutic approaches have been directed at hormonal therapy. The mostcommon therapy involves the use of Danazol. Danazol is a syntheticsteroid with androgenic action suppressing the pituitary gland cyclingnecessary for menstrual periods. Amenorrhea, or lack of menstrualperiods results. Though providing some relief from the pain ofendometriosis adverse side effects are experienced in up to 80% of womenusing Danazol (Greenblatt R B et al., 1971, “Clinical studies with ananti-gonadotropin—danazol”, Fertil Steril 22:102). Notably these sideeffects include weight gain, fluid retention, acne, decreased breastsize, hot flushes and mood changes. In addition to Danazol, otherhormonal manipulations used in the management of endometriosis involveuse of gonadotropin releasing hormone analogues (GnRH) and the druggestrinone, a synthetic steroid derived from 19-nortestosterone. Theside effects associated with these therapies are significant and includethe spectrum of symptoms associated with hypoestrogenism and menopause.These include hot flushes, night sweats, and osteoporosis (Telimaa E Jet al., 1987, “Placebo-controlled comparison of danazol and high-dosemedroxyprogesterone acetate in the treatment of endometriosis”, GynecolEndocrinol 1:51; Thomas E. J. et al., 1987, “Impact of gestrinone on thecourse of asymptomatic endometriosis”, Br. Med J., 294:272). Clearly,more benign approaches to the management of the pain of endometriosisare needed.

[0012] 2.4. Cervical Dysplasia

[0013] Cervical dysplasia, also known as cervical intraepithelialneoplasia (CIN), refers to the occurrence of abnormal cells and tissueinvolving the surface of the uterine cervix, which consists of thelowest part of the uterus. Anatomically, the cervix provides the portalof entry from the vagina to the interior of the uterus. Its surfaceconsists of an epithelial “transition-zone” where superficial, flattenedcells characteristic of vaginal mucosa abruptly change to cuboidalepithelial cells characteristic of the interior surface of the uterus(endometrium). The designations CIN I, II and III refer to mild,moderate, and severe dysplasia/carcinoma in situ (CIS) of the cervicalepithelium, respectively. This histological grading scheme is basedlargely on the extent to which the thickness of the epithelium isreplaced by abnormal, mitotically active cells with enlarged,hyperchromatic nuclei (Ferenczy A et al., “Cervical IntraepithelialNeoplasia and Condyloma”, In R. J. Kurman, Editor. Blaustein's Pathologyof the Female Genital Tract, Springer-Verlag, 1987). Untreated, a subsetof dysplastic lesions will advance to cervical cancer, with a frequencythat increases dramatically in CIN III lesions (Ostor A G, 1993, Int. J.Gynecol. Path. 12:186-192).

[0014] In many developing countries, cervical cancer is the most commoncancer (excluding skin) in women and the major cause of cancer-relateddeaths in women (Parkin D et al., 1993, Int J Cancer 54:594-606). In theUnited States, abnormalities on Papanicolaou, or “Pap” smears aredetected in millions of women annually, resulting in an estimated annualcost of $6 billion for patient evaluation and treatment (Kurman R etal., 1994, JAMA 271:1866-9).

[0015] Cervical dysplasia in women is a common condition that is linkedto infection by HPV (Walboomers J M et al., 1999, “Human papillomavirusis a necessary cause of invasive cervical cancer worldwide”, J Pathol.189(1):12-9). While dependent on the presence of HPV, the occurrence ofcervical dysplasia in women has not been related to estrogen orprogesterone levels. Regarding estrogen metabolism, the severity ofcervical dyplasia, documented by stage of CIN on biopsy, has beenassociated with a reduced 2-hydroxy metabolism of estrogen in some women(Sepkovic et al., 1995, “Estrogen Metabolite Ratios and Risk Assessmentof Hormone-related Cancers”, Annals New York Academy of Sciences768:312-316).

[0016] The most common methods used to treat cervical dysplasia includeremoval or eradication of the abnormal tissue, using methods such ascryosurgery, laser vaporization, and surgical removal. Due to thepotential for harm to normal tissue, and recurrence of dysplasiafollowing surgery, a need clearly exists for more effective and lessinvasive therapy.

[0017] 2.5. Dietary Indoles

[0018] 3,3′-Diindolylmethane (DIM), as well as its precursor,Indole-3-carbinol (I3C), and cogener,2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTr-1), are naturalphytochemicals and part of the family of dietary indoles discovered incruciferous vegetables. DIM and I3C are found in cruciferous vegetablesincluding broccoli, cauliflower, cabbage and Brussels sprouts (BradfieldC A and Bjeldanes L F, 1987, “High performance liquid chromatographicanalysis of anticarcinogenic indoles in Brassica oleracea”, J. Agric.Food Chem. 35:46-49). DIM, together with the linear trimer, LTr-1, areformed after the enzymatic release of I3C from parent glucosinolatesfound in all cruciferous vegetables.

[0019] It is now known that there is a connection between dietarycruciferous indoles and estrogen metabolism. H. Leon Bradlow, Ph.D. andhis group at the Strang Cancer Prevention Laboratory in New York werethe first to establish the link between phytonutrients from cruciferousvegetables and estrogen metabolism. They showed that supplemental use ofI3C can act to promote a dramatic change in the metabolism of estrogen(Michnovicz J J et al., 1997, “Changes in levels of urinary estrogenmetabolites after oral indole-3-carbinol treatment in humans”, J NatlCancer Inst. 89(10):718-23). This change in metabolism has the power togreatly reduce estrogen exposure as a risk for cancer and provides adietary approach to improving estrogen metabolism. I3C has been shown toincrease 2-hydroxy estrogen metabolism in women (Bradlow H L et al.,1994, “Long-term responses of women to indole-3-carbinol or a high fiberdiet”, Cancer Epidemiol Biomarkers Prev. 3(7):591-5). When cruciferousphytochemicals are added to the diet, estrogen metabolism is shifted.This produces a predominance of 2-hydroxy and 2-methoxyestrogens(Michnovicz J J et al., 1997, “Changes in levels of urinary estrogenmetabolites after oral indole-3-carbinol treatment in humans”, J NatlCancer Inst. 89(10):718-23). An increased proportion of 2-hydroxymetabolites is correlated to protection from breast cancer. Thisrelationship has been documented in case-control studies (Ho G H et al.,1998, “Urinary 2/16 alpha-hydroxyestrone ratio: correlation with seruminsulin-like growth factor binding protein-3 and a potential biomarkerof breast cancer risk”, Ann Acad Med Singapore 27:294-299; Schneider J.et al., 1982, “Abnormal oxidative metabolism of estradiol in women withbreast cancer”, Proc Natl Acad Sci USA 79:3047-3051). The 2-hydroxymetabolites have been called “good estrogens” (Bradlow H L et al., 1996,“2-hydroxyestrone: the ‘good’ estrogen”, J Endocrinol. 150Suppl:S259-65), and may function as antioxidants (Komura S et al., 1996,“Catecholestrogen as a natural antioxidant”, Ann N Y Acad Sci.786:419-429).

[0020] With regard to prior art and the dietary indoles, thesupplemental use of I3C, which converts in part to DIM, LTr-1, and othercompounds after passage through the stomach, has been the subject ofU.S. Pat. No. 5,895,787, describing the use of I3C and related dietaryindoles to reduce the symptoms of fibromyalgia. Despite this use, norelationship between fibromyalgia and estrogen status has beendocumented (Bengtsson A. and Henriksson, K. G., 1986, “Primaryfibromyalgia. A clinical and laboratory study of 55 patients”, Scand J.Rheumatol 15(3):340-7). Published reports have demonstrated someclinical improvement following dietary supplementation with I3C inrecurrent laryngeal papillomatosis and cervical dysplasia (Rosen C A etal., 1998, “Preliminary results of the use of indole-3-carbinol forrecurrent respiratory papillomatosis”, Otolaryngology Head Neck Surgery118: 810-5; Jin L. et al., 1999, “Indole-3-carbinol prevents cervicalcancer in human papilloma virus type 16 (HPV16) transgenic mice”, CancerRes. 59(16):3991-7; Baugh S M et al., 1998, “Treatment of cervicaldysplasia with indole-3-carbinol”, Ray A. Barlow Scientific Symposium,Louisiana State University Medical School, Shreveport, La.; Bell M C etal., 2000, “Placebo-controlled trial of indole-3-carbinol in thetreatment of CIN”, Gynecol Oncol. 78(2):123-9). These diseases are bothconditions related to the presence of the human papilloma virus. Reportsof adverse side effects with I3C use in animals and in at higher dosesin individuals with respiratory HPV infection (Recurrent RespiratoryPapillomatosis [“RRP”]) have discouraged further clinical testing of I3C(Rosen C A et al., 1998, “Preliminary results of the use ofindole-3-carbinol for recurrent respiratory papillomatosis”,Otolaryngology Head Neck Surgery 118:810-5; Wilker C et al., 1996,“Effects of developmental exposure to indole-3-carbinol or2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of malerat offspring”, Toxicol Appl Pharmacol. 141(1):68-75). I3C's use isassociated with a number of safety concerns in reproductive-age womendue to its enzyme-inducing actions and developmental toxicity.

[0021] Thus, the individual metabolic products of I3C are beinginvestigated to discover safer and more effective alternatives to I3C.Following oral administration of I3C, DIM is but one of many derivativesgenerated from I3C. I3C is highly reactive in stomach acid, which leadsto the production of a large number of biologically active byproductsdistinct from DIM. DIM arises from I3C as one of the reaction productsin more neutral pH environments, not typically seen in the gastricenvironment. I3C exposure to acid produces many non-DIM products,including indolo(3,2,b)carbazole (ICZ), and the cyclic trimer,5,6,11,12,17,18-hex-hydrocyclononal[1,2-b:4,5-b′:7,8-b]:triindole, andothers which are more highly enzyme inductive than DIM (De Kruif C A etal., 1991, “Structure elucidation of acid reaction products ofindole-3-carbinol: detection in vivo and enzyme induction in vitro”,Chem Biol Interact 80(3):303-15).

[0022] DIM differs from I3C in its structure and in its chemical andbiological properties. Unlike I3C, DIM is an acid-stable indole withless influence on carcinogen metabolism than I3C and non-DIM productsfrom I3C (Bradfield C A and Bjeldanes L F, 1987, “Structure-activityrelationships of dietary indoles: a proposed mechanism of action asmodifiers of xenobiotic metabolism”, J Toxicol Environ Health21(3):311-23). Unlike I3C, DIM has been shown to influence estrogenmetabolism in animals without the need for intra-gastric conversion intoother indole reaction products (Jellinck P H et al., 1993, “Ah receptorbinding properties of indole carbinols and induction of hepaticestradiol hydroxylation”, Biochem Pharmacol 45:1129-1136).

[0023] In cell culture, DIM has been shown to have both growth promotingand growth inhibitory properties. In MCF7 breast cancer cells, DIMpromoted growth when tested in the absence of estrogen (Riby J E et al.,2000, “Ligand-independent activation of estrogen receptor function by3,3′-diindolylmethane in human breast cancer cells”, Biochem Pharmacol60(2):167-77). When tested in colon cancer cells, DIM demonstratedselective growth inhibitory activity which was not observed innon-cancerous colon cells (Gamet-Payrastre L et al., 1998, “Selectivecytostatic and cytotoxic effects of glucosinolates hydrolysis productson human colon cancer cells in vitro”, Anti-Cancer Drugs 9:141-148).

[0024] In another study, oral DIM was shown to impede the growth ofcarcinogen-induced breast cancer in animals (Chen I. et al., 1998, “Arylhydrocarbon receptor-mediated antiestrogenic and antitumorigenicactivity of diindolylmethane”, Carcinogenesis 19(9):1631-9).

[0025] There have been no reports on the usefulness of DIM or LTr-1 inthe treatment or prevention of mastalgia, endometriosis or HPV-relatedconditions, including cervical dysplasia. Isolation and formulationrequirements for DIM as a pure substance limited its availability foranimal experiments. Pure, isolated DIM was not available for human useprior to Summer, 1998. Use of DIM and LTr-1 in absorption enhancingformulations for improving the balance of estrogen metabolites has beenthe subject of earlier investigation by the present inventor andprovides the basis of U.S. Pat. No. 6,086,915. This work has allowed forthe present investigation of the use of dietary indoles as dietarysupplements to beneficially impact mastalgia, endometriosis, andcervical dysplasia.

[0026] Therefore, safer and more consistently effective treatments areneeded for mastalgia, endometriosis and HPV-related conditions,including cervical dysplasia.

3. SUMMARY OF THE INVENTION

[0027] The present invention relates to compositions and methods forprevention or reduction of symptoms associated with mastalgia,endometriosis and HPV-related diseases including, but not limited to,cervical dysplasia, by administering phytochemicals, e.g., dietaryindoles. In a preferred embodiment, the pain associated withendometriosis and mastalgia is prevented or reduced. In anotherembodiment, the presence of a marker associated with endometriosis isreduced through phytochemical treatment. In yet another preferredembodiment, the progression of cervical dysplasia to cervical cancer isprevented or cervical dysplasia is reduced and/or eliminated. Among thephytochemicals useful in the compositions and methods of the inventionare diindolylmethane (DIM), and its cogener,2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTr-1).

[0028] Also according to the present invention, a pharmaceuticalcomposition is provided, which comprises a phytochemical, preferably DIMand/or LTr-1, and, optionally, pharmaceutically acceptable carriers.

[0029] In one aspect, the present invention provides a method oftreating cervical dysplasia in a subject having cervical dysplasiacomprising administering to the subject an amount of a dietary indoleselected from the group consisting of DIM and LTr-1 effective to reduceone or more symptoms associated with cervical dysplasia. In a preferredembodiment, the dietary indole is DIM. In a more preferred embodiment,DIM is suspended as microparticles in a starch carrier matrix. In otherembodiments, the dietary indole is formulated as a cream or suppository.In certain embodiments, the dietary indole is administered by directapplication to the vaginal or cervical mucosa of the subject. In certainembodiments, the dietary indole is formulated as a cream, and isadministered transdermally. In certain embodiments, the dietary indoleis administered orally.

[0030] In another aspect, the present invention provides a method ofpreventing cervical dysplasia in a subject in danger of developingcervical dysplasia comprising administering to the subject an amount ofa dietary indole selected from the group consisting of DIM and LTr-1effective to prevent one or more symptoms associated with cervicaldysplasia. In a preferred embodiment, the dietary indole is DIM. In amore preferred embodiment, DIM is suspended as microparticles in astarch carrier matrix. In other embodiments, the dietary indole isformulated as a cream or suppository. In certain embodiments, thedietary indole is administered by direct application to the vaginal orcervical mucosa of the subject. In certain embodiments, the dietaryindole is formulated as a cream, and is administered transdermally. Incertain embodiments, the dietary indole is administered orally.

4. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention is based upon the observation thatadministration of phytochemicals, in particular, DIM, and LTr-1, hasimproved symptoms of mastalgia, endometriosis, and HPV-relatedconditions, including cervical dysplasia. In particular, theadministration of DIM, either topically and orally, is followed by atherapeutic response in mastalgia and cervical dysplasia.

[0032] The facilitated delivery of DIM and related indoles as dietarysupplements may be accomplished according to formulations and methodsdescribed in U.S. Pat. No. 6,086,915. The effectiveness of supplementalDIM is further supported by co-administration of phytochemicals (e.g.,DIM, LTr-1) with grapefruit concentrate, which additionally facilitatesthe absorption of phytochemicals (e.g., DIM, LTr-1).

[0033] 4.1. Methods of Treating Mastalgia

[0034] The invention provides compositions and methods for prevention orreduction of symptoms associated with mastalgia. In a preferredembodiment, breast pain of subjects suffering from mastalgia isprevented, reduced and/or eliminated through the administration ofphytochemicals, e.g., dietary indoles, in a pharmaceutically acceptablefashion. In preferred embodiments, the phytochemicals are DIM, I3C andLTr-1. In particular embodiments, I3C, DIM, or LTr-1, alone or incombination with each other or other dietary supplements, areadministered orally in, for example, the form of encapsulated dietarysupplements.

[0035] The I3C is preferably administered at a dose of 200-500 mg perday. In alternative embodiments, I3C is administered at doses of 200-300mg per day, 300-400 mg per day and 400-500 mg per day.

[0036] DIM is administered providing 150-500 mg per day of DIM. Inpreferred embodiments, the dose of DIM, I3C or LTr-1 is 150-200 mg perday, 200-300 mg per day, 300-400 mg per day, and 400-500 mg per day.

[0037] In a preferred embodiment, DIM is administered in an absorptionenhancing formulation, as described in U.S. Pat. No. 6,086,915,providing 60-500 mg per day of DIM suspended as microparticles in astarch carrier matrix. In preferred embodiments, the dose of processedDIM is 60-100 mg per day, 100-200 mg per day, 200-300 mg per day,300-400 mg per day, and 400-500 mg per day.

[0038] The LTr-1 is preferably administered in an absorption enhancingformulation providing 60-500 mg per day of LTr-1 suspended asmicroparticles in a starch carrier matrix as previously described,however, the present invention contemplates the administration of anypreparation of LTr-1. In a preferred embodiment, the dose of LTr-1 is150-200 mg per day. In preferred embodiments, the dose of processedLTr-1 is 60-100 mg per day, 200-300 mg per day, 300-400 mg per day, and400-500 mg per day.

[0039] Doses of the phytochemicals of the invention can also becalculated based upon the body weight of the subject to be treated.Doses of phytochemicals between 0.5 and 3 mg per kg of body weight perday are preferred. In another preferred embodiment, the phytochemicalsare administered at a dose of between 0.5 and 2.0 mg per kg per day,preferably 1.5 mg per kg per day.

[0040] Alternatively, the co-administration of grapefruit, grapefruitconcentrate, grapefruit juice, or grapefruit juice concentrate, or othergrapefruit-derived composition along with a dietary indole (e.g., I3C,DIM or LTr-1) can be used to increase absorption of the phytochemicalsand promote even more efficient relief from the symptoms of mastalgia.

[0041] In an alternative embodiment, the dietary indole (e.g., DIM orLTr-1) is administered in the form of a liposomal sublingual sprayapplied directly to the oral mucosa. This liposomal suspension providesphytochemical loaded liposomes to administer the phytochemicals andcreate a sustained delivery system. Dietary indole (e.g., DIM or LTr-1)containing liposomes are sequestered in the oral mucosa, allowingabsorption which avoids “first pass” hepatic metabolism. The liposomalspray uses standard liposomal preparation and structural lipidingredients (Ranade V V, 1989, “Drug delivery systems. 1. Site-specificdrug delivery using liposomes as carriers”, J. Clin. Pharmacol.29(8):685-94). In a preferred embodiment, the liposomal spray isadministered at a dose of 5-30 mg of dietary indole (e.g., DIM or LTr-1)daily delivered in 2-12 sprays of a typical liposomal preparation.

[0042] Alternatively the phytochemicals (e.g., DIM or LTr-1) may beadministered in the form of a transdermal cream applied directly to theskin. This cream utilizes various absorption enhancing emollients andconsists of phytochemical (e.g., DIM or LTr-1) in a concentration of1-3% by weight. It is designed as a moisturizing cosmetic that isformulated to allow application directly to painful breasts in women notwishing to take phytochemicals orally. Formulations are also made withthe neurohormone, melatonin, to provide a nighttime cosmetic mosturizeroffering the benefits of melatonin in combination with the phytochemical(e.g., DIM or LTr-1). This allows application of cruciferous indoles andmelatonin directly to underlying breast tissue with the added benefit ofsleep regulating action from melatonin. In a particular embodiment,application of from 5-10 cc of the transdermal preparation daily is usedto administer from 5-30 mg of DIM or other dietary indole per day, andoptionally, 3-10 mg of melatonin per day.

[0043] Alternatively, the phytochemical (e.g., DIM or LTr-1) may beadministered in the form of a vaginal cream or suppository containingmicrocrystalline phytochemical (e.g., DIM or LTr-1) in a combined doseof 20-100 mg. This allows application of cruciferous indoles directly tovaginal and cervical mucosa for the benefit of cervical dysplasia.

[0044] The phytochemicals of the invention may be administered in anyappropriate amount in any suitable galenic formulation and following anyregime of administration.

[0045] The actual administered amount of phytochemical may be decided bya supervising physician and may depend on multiple factors, such as, theage, condition, file history, etc., of the patient in question.

[0046] The subject, or patient, to be treated using the methods of theinvention is an animal, e.g., a mammal, and is preferably human, and canbe a fetus, child, or adult. In a preferred embodiment, the subject is ahuman female.

[0047] 4.2. Methods of Treating Endometriosis

[0048] The invention provides compositions and methods for reduction orprevention of symptoms associated with endometriosis. In a preferredembodiment, the pain of subjects suffering from endometriosis isprevented, reduced and/or eliminated through the administration ofphytochemicals in a pharmaceutically acceptable fashion. In anotherpreferred embodiment, the levels of an endometriosis marker (e.g.,Ca-125 antigen, a serum marker of endometriosis) in subjects sufferingfrom endometriosis is lowered through the administration ofphytochemicals in a pharmaceutically acceptable fashion. In preferredembodiments, the phytochemicals are DIM, I3C and LTr-1. In particularembodiments, DIM, or LTr-1, alone or in combination with each other orother dietary supplements, are administered orally in, for example, theform of encapsulated dietary supplements.

[0049] DIM is administered providing 30-500 mg per day of DIM. Inpreferred embodiments, the dose of DIM, I3C or LTr-1 is 30-100 mg perday, 100-200 mg per day, 200-300 mg per day, 300-400 mg per day, and400-500 mg per day.

[0050] In a preferred embodiment, DIM is administered in an absorptionenhancing formulation, as described in U.S. Pat. No. 6,086,915,providing 30-500 mg per day of DIM suspended as microparticles in astarch carrier matrix. In preferred embodiments, the dose of processedDIM is 30-100 mg per day, 100-200 mg per day, 200-300 mg per day,300-400 mg per day, and 400-500 mg per day.

[0051] The LTr-1 is preferably administered in an absorption enhancingformulation providing 30-400 mg per day of LTr-1 suspended asmicroparticles in a starch carrier matrix as previously described,however, the present invention contemplates the administration of anypreparation of LTr-1. In a preferred embodiment, the dose of LTr-1 is100-200 mg per day. In preferred embodiments, the dose of processedLTr-1 is 30-100 mg per day, 200-300 mg per day, 300-400 mg per day, and400-500 mg per day.

[0052] Doses of the phytochemicals of the invention can also becalculated based upon the body weight of the subject to be treated.Doses of phytochemicals between 1 and 3 mg per kg of body weight per dayare preferred. In another preferred embodiment, the phytochemicals areadministered at a dose of between 1.5 and 2.5 mg per kg per day,preferably 2.0 mg per kg per day.

[0053] Alternatively, the co-administration of grapefruit, grapefruitconcentrate, grapefruit juice, or grapefruit juice concentrate, or othergrapefruit-derived composition along with I3C, DIM or LTr-1 can be usedto increase absorption of the phytochemicals and promote even moreefficient relief from the symptoms of endometriosis, including thereduction of markers associated with endometriosis.

[0054] In an alternative embodiment, the phytochemical (e.g., DIM orLTr-1) is administered in the form of a liposomal sublingual sprayapplied directly to the oral mucosa. This liposomal suspension providesphytochemical loaded liposomes to administer the phytochemicals andcreate a sustained delivery system. DIM and LTr-1 containing liposomesare sequestered in the oral mucosa, allowing absorption which avoids“first pass” hepatic metabolism. The liposomal spray uses standardliposomal preparation and structural lipid ingredients (Ranade V V,1989, “Drug delivery systems. 1. Site-specific drug delivery usingliposomes as carriers,” J. Clin. Pharmacol. 29(8):685-94; Crommelin D JA and Schreir H, “Liposomes”, Colloidal Drug Delivery Systems, Kreuter,J. editor, Marcel Dekker, N.Y., 1994, p. 85). In a preferred embodiment,the liposomal spray is administered at a dose of 5-30 mg ofphytochemical daily delivered in 2-12 sprays of a typical liposomalpreparation.

[0055] Alternatively the phytochemicals (e.g., DIM or LTr-1) may beadministered in the form of a transdermal cream applied directly to theskin. This cream utilizes various absorption-enhancing emollients andconsists of DIM or LTr-1 in a concentration of 1-3% by weight. It isdesigned as a moisturizing cosmetic which is formulated to allowapplication directly to the skin of women not wishing to takephytochemicals orally. Formulations are also made with the neurohormone,melatonin, to provide a nighttime cosmetic moisturizer offering thebenefits of melatonin in combination with the phytochemical (e.g., DIMor LTr-1). This provides the added benefit of sleep regulating actionfrom melatonin. In a particular embodiment, application of from 5-10 ccof the transdermal preparation daily is used to administer from 5-30 mgof phytochemical (e.g., DIM or LTr-1) per day, and optionally, 3-10 mgof melatonin per day.

[0056] Alternatively the phytochemical (e.g., DIM or LTr-1) may beadministered in the form of a vaginal cream or suppository containingmicrocrystalline DIM or LTr-1 in a combined dose of 20-100 mg. Thisallows application of cruciferous indoles directly to vaginal andcervical mucosa for the benefit of cervical dysplasia.

[0057] The phytochemicals of the invention may be administered in anyappropriate amount in any suitable galenic formulation and following anyregime of administration.

[0058] The actual administered amount of phytochemical may be decided bya supervising physician and may depend on multiple factors, such as, theage, condition, file history, etc., of the patient in question.

[0059] The subject, or patient, to be treated using the methods of theinvention is an animal, e.g., a mammal, and is preferably human, and canbe a fetus, child, or adult. In a preferred embodiment, the subject is ahuman female.

[0060] 4.3. Methods of Treating HPV-Related Conditions, IncludingCervical Dysplasia

[0061] The invention provides compositions and methods for prevention orreduction of symptoms associated with cervical dysplasia, a knownHPV-related condition. In a preferred embodiment, the progression ofcervical dysplasia to cervical cancer is prevented or cervical dysplasiais reduced and/or eliminated through the administration ofphytochemicals in a pharmaceutically acceptable fashion. In preferredembodiments, the phytochemicals are DIM and LTr-1. In particularembodiments, DIM, or LTr-1, alone or in combination with each other orother dietary supplements, are administered orally in, for example, theform of encapsulated dietary supplements.

[0062] DIM is administered providing 30-500 mg per day of DIM. In apreferred embodiment, DIM is administered providing 150-500 mg per dayof DIM. In more preferred embodiments, the dose of DIM or LTr-1 is30-100 mg per day, 100-200 mg per day, 150-200 mg per day, 200-300 mgper day, 300-400 mg per day, and 400-500 mg per day.

[0063] In a preferred embodiment, DIM is administered in an absorptionenhancing formulation, as described in U.S. Pat. No. 6,086,915,providing 30-500 mg per day of DIM, preferably 60-500 mg per day, of DIMsuspended as microparticles in a starch carrier matrix. In preferredembodiments, the dose of processed DIM (25% DIM by weight of theformula) is 30-100 mg per day, 60-100 mg per day, 100-200 mg per day,200-300 mg per day, 300-400 mg per day, and 400-500 mg per day. In morepreferred embodiments, the dose of processed DIM (25% DIM by weight ofthe formula) is 120-400 mg per day, 240-400 mg per day, 400-800 mg perday, 800-1,200 mg per day, 1,200-1600 mg per day, and 1,600-2,000 mg perday.

[0064] The LTr-1 is preferably administered in an absorption enhancingformulation providing 30-500 mg per day, preferably 60-500 mg per day,of LTr-1 suspended as microparticles in a starch carrier matrix aspreviously described, however, the present invention contemplates theadministration of any preparation of LTr-1. In a preferred embodiment,the dose of LTr-1 is 100-200 mg per day, more preferably 150-200 mg perday. In preferred embodiments, the dose of processed LTr-1 is 30-100 mgper day, 60-100 mg per day, 200-300 mg per day, 300-400 mg per day, and400-500 mg per day. In a preferred embodiment, the dose of LTr-1 is100-200 mg per day, more preferably 150-200 mg per day. In preferredembodiments, the dose of processed LTr-1 is 120-400 mg per day, 240-400mg per day, 800-1,200 mg per day, 1,200-1600 mg per day, and 1,600-2000mg per day.

[0065] Doses of the phytochemicals of the invention can also becalculated based upon the body weight of the subject to be treated.Doses of phytochemicals between 0.5 and 5 mg per kg of body weight perday are preferred. Doses of phytochemicals between 1 and 3 mg per kg ofbody weight per day are preferred. In another preferred embodiment, thephytochemicals are administered at a dose of between 1.5 and 3.0 mg perkg per day, more preferably between 1.5 and 2.5 mg per kg per day,preferably 1.5 or 2.0 mg per kg per day.

[0066] Alternatively, the co-administration of grapefruit, grapefruitconcentrate, grapefruit juice, or grapefruit juice concentrate, or othergrapefruit-derived composition along with a dietary indole (e.g., DIM orLTr-1) can be used to increase absorption of the phytochemicals andpromote even more efficient relief from the symptoms of cervicaldysplasia.

[0067] In an alternative embodiment, the dietary indole (e.g., DIM orLTr-1) is administered in the form of a liposomal sublingual sprayapplied directly to the oral mucosa. This liposomal suspension providesphytochemical loaded liposomes to administer the phytochemicals andcreate a sustained delivery system. Dietary indole (e.g., DIM or LTr-1)containing liposomes are sequestered in the oral mucosa, allowingabsorption which avoids “first pass” hepatic metabolism. The liposomalspray uses standard liposomal preparation and structural lipidingredients (Ranade V V, 1989, “Drug delivery systems. 1. Site-specificdrug delivery using liposomes as carriers,” J. Clin. Pharmacol.29(8):685-94). In a preferred embodiment, the liposomal spray isadministered at a dose of 5-30 mg of dietary indole (e.g., DIM or LTr-1)daily delivered in 2-12 sprays of a typical liposomal preparation. In apreferred embodiment, the liposomal spray is administered at a dose of15-130 mg of dietary indole (e.g., DIM or LTr-1) daily delivered in 2-12sprays of a typical liposomal preparation.

[0068] Alternatively the phytochemicals (e.g., DIM or LTr-1) may beadministered in the form of a transdermal cream or suppository applieddirectly to the skin, genitalia, or cervical epithelium. These topicalformulations utilize various absorption enhancing emollients and consistof phytochemical (e.g., DIM or LTR) in a concentration of 1-3%, morepreferably 1-2% by weight. This allows application of cruciferousindoles, DIM or LTR, directly to underlying cervical tissue. In aparticular embodiment, application of from 5 to 10 cc of the transdermalpreparation daily is used to administer from 5-30 mg of DIM per day tothe skin. In a particular embodiment, application of from 5 to 10 cc ofthe transdermal preparation daily is used to administer from 50-100 mgof DIM per day to the skin.

[0069] Alternatively, the phytochemical (e.g., DIM or LTr-1) may beadministered in the form of a vaginal cream or suppository containingmicrocrystalline phytochemical (e.g., DIM or LTr-1) in a combined doseof 20-100 mg per day, or 20-200 mg per day, more preferably 50-100 mgper day. This allows application of cruciferous indoles directly tovaginal and cervical mucosa for the benefit of cervical dysplasia.

[0070] The phytochemicals of the invention may be administered in anyappropriate amount in any suitable galenic formulation and following anyregime of administration.

[0071] The actual administered amount of phytochemical may be decided bya supervising physician and may depend on multiple factors, such as, theage, condition, file history, etc., of the patient in question.

[0072] The subject, or patient, to be treated using the methods of theinvention is an animal, e.g., a mammal, and is preferably human, and canbe a fetus, child, or adult. In a preferred embodiment, the subject is ahuman female. 4.4. Pharmaceutical Compositions

[0073] The pharmaceutical compositions according to the presentinvention preferably comprise one or more pharmaceutically acceptablecarriers and the active constituents. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipient thereof.

[0074] It will be appreciated that the amounts of phytochemical requiredfor said treatment will vary according to the route of administration,the disorder to be treated, the condition, age, and file history of thesubject, the galenic formulation of the pharmaceutical composition, etc.

[0075] Preferably, the phytochemical used in the invention has beenprocessed to enhance bioavailability, as is described in U.S. Pat. No.6,086,915. So processed DIM or LTr-1 is referred to as “processed DIM”and “processed LTr-1”, respectively. However, any suitable preparationof phytochemical can be used in the methods and compositions of theinvention.

[0076] The following is a list of ingredients useful for formulatingprocessed DIM or LTr-1:

[0077] 1. About 10 to about 40 percent by weight of LTr-1 or DIM.

[0078] 2. About 10 to about 40 percent by weight of the following, aloneor in combination: vitamin E succinate polyethylene glycol 1000; vitaminE succinate Polyethylene glycols with polyethylene glycol (with amolecular weight range of 400-2000); other polyethylene glycol esterssuch as those formed by fatty acids such as oleic acid or stearic acid;polyvinylpyrrolidones; polyvinylpolypyrrolidones; Poloxamer 188, Tweens;or Spans.

[0079] 3. About 5 to about 20 percent by weight of the following, aloneor in combination: phosphatidyl choline (derived from soy lecithin andsupplied as Phospholipon 50G from Rhone Poulenc Rorer); dioleoylphosphatidylcholine; phoshatidylglycerol; dioleoylphosphatidylglycerol;dimyristoylphosphatidylcholine; dipalmitoylphosphatidylcholine;phosphatidylethalolamines; phosphatidylserines; or sphingomyelins; orother sources of phospholipids as those from purified Milk Fat GlobuleMembrane; glycerolesters; poly glycerol esters; or ethoxylated castoroil.

[0080] 4. About 15 to about 30 percent by weight of the following, aloneor in combination: hexanol; ethanol; butanol; heptanol;2-methyl-1-pentanol; various ketone solvents that would be acceptable infoods such as methyl ethyl ketone, acetone and others; propylene glycol;and certain ester solvents such as ethyl acetate.

[0081] 5. About 20 to about 40 percent by weight of the following, aloneor in combination: modified starch such as Capsul™ Starch from NationalStarch, Inc.; methylcellulose; hydroxypropyl methylcellulose;hydroxyethylcellulose; hydroxypropylethylcellulose; pectin; gum arabic;gelatin; or other polymeric matrix-forming preparation known to thoseskilled in the art, soluble in water and, suitable for spray drying.

[0082] 6. About 0.5 to about 35 percent by weight of the following,alone or in combination: aerosil 200; or any other flow enhancingexcipient from silica, or related salt, known to those skilled in theart.

[0083] The following is a detailed method of formulating processed DIM:

[0084] 1. 6.75 kg of TPGS is heated just beyond its melting point withconstant stirring in a heated vessel (“First vessel”).

[0085] 2. 9.38 kg of hexanol and 9.83 kg of jet milled DIM is added tothe first vessel and the mixture stirred to a uniform suspension at 70°C. 1.4 kg of phosphatidyl choline is then added.

[0086] 3. In a second larger vessel, 185 L of water and 10.7 kg ofstarch are thoroughly mixed with a Cowles blade. This mixture isneutralized to pH 7 with a small amount of sodium carbonate and thenheated to 75° C. and stirred for 1 hour.

[0087] 4. The contents of the first vessel is added to the starchmixture in the second larger vessel and thoroughly mixed with ahomogenizing rotor/stator type mixer at moderate speed for 15 minutes.

[0088] 5. The mixture from step 4 is spray dried with a small amount(approximately 0.5%) of hydrophilic silica to provide a free flowingpowder of finely dispersed microparticles containing the co-precipitatedTPGS, phosphatidyl choline and DIM in an amorphous, non-crystallinestructure.

[0089] 6. The flowable powder product is collected and stored inevacuated foil sacks, after de-aerating and flushing with nitrogen.

[0090] 7. Analysis of presence of unchanged dietary ingredient, revealsabout 30 to about 33 percent by weight of DIM.

[0091] The procedure of making processed DIM may be summarized asfollows:

[0092] (a) heating one or more solubilizing emulsifiers selected fromthe group consisting of vitamin E succinate polyethylene glycol 1000,polyvinylpyrrolidone, polyoxyethylene stearate, sodium cholate,deoxycholate and taurocholate;

[0093] (b) adding to the product of step (a) a solvent and a surfactantphospholipid selected from the group consisting of phosphatidyl choline,dioleoyl phosphatidyl choline, phosphatidylglycerol,dioleoylphosphatidylglycerol, dimyristoylphosphatidylcholine,dipalitoylphosphatidylcholine, phosphatidylethanolamine,phosphatidylserine and sphingomyelin to produce a solution;

[0094] (c) dissolving in the solution of step (b) LTr-1 and/or DIM;

[0095] (d) adding to the solution of step (c) a solution containing anencapsulator;

[0096] (e) mixing the solution produced in step (d) to produce amicrodispersion with a particle size of 5 microns or less; and

[0097] (f) spray drying the resulting mixture to leave a solidhydrophobic phytochemical composition.

[0098] In general, a suitable (therapeutically effective) amount of I3Cis 300-500 mg per day. In general, a suitable (therapeuticallyeffective) amount of DIM or LTR is 100-500 mg per day. DIM is preferablyadministered in an absorption enhancing formulation, as described inU.S. Pat. No. 6,086,915, at 50-200 mg per day, more preferably 400-800mg per day, as a suspension of microparticles in a starch carriermatrix. The LTr-1 is preferably administered in an absorption enhancingformulation at 50-200 mg per day, more preferably 200-800 mg per day, asa suspension of microparticles in a starch carrier matrix. The actuallyadministered amounts of phytochemical may be decided by a supervisingphysician. The phytochemicals of the invention may be administered aloneor in combination with one another, and/or with other dietarysupplements. The combinations of phytochemicals and supplements can bein the same composition for administering in combination concurrently,or in different compositions for administering concurrently butseparately, or sequentially.

[0099] Therapeutic formulations include those suitable for parenteral(including intramuscular and intravenous), oral, rectal, intra-vaginal,or intradermal administration, although oral administration is thepreferred route. Thus, the pharmaceutical composition may be formulatedas tablets, pills, syrups, capsules, suppositories, formulations fortransdermal application, formulations for intradermal uses, powders,especially lyophilized powders for reconstitution with a carrier forintravenous administration, etc.

[0100] The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; asweetening agent, such as sucrose or saccharin; and/or a flavoringagent, such as peppermint, methyl salicylate, or orange flavoring.

[0101] Therapeutic formulations suitable for oral administration, e.g.,tablets and pills, may be obtained by compression or molding, optionallywith one or more accessory ingredients. Compressed tablets may beprepared by mixing phytochemicals, and compressing this mixture in asuitable apparatus into tablets having a suitable size. Prior to themixing, the phytochemical may be mixed with a binder, a lubricant, aninert diluent and/or a disintegrating agent.

[0102] In a preferred embodiment, phytochemical is mixed with a binder,such as microcrystalline cellulose, and a surfactant, such as sodiumlauryl sulphate until a homogeneous mixture is obtained. Subsequently,another binder, such as polyvidone, is transferred to the mixture understirring with a small amount of added water. This mixture is passedthrough granulating sieves and dried by desiccation before compressioninto tablets in a standard tableting apparatus.

[0103] A tablet may be coated or uncoated. An uncoated tablet may bescored. A coated tablet may be coated with sugar, shellac, film or otherenteric coating agents.

[0104] Therapeutic formulations suitable for parenteral administrationinclude sterile solutions or suspensions of the active constituents. Anaqueous or oily carrier may be used. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Formulations for parenteraladministration also include a lyophilized powder comprisingphytochemical that is to be reconstituted by dissolving in apharmaceutically acceptable carrier that dissolves said phytochemical.

[0105] When the pharmaceutical composition is a capsule, it may containa liquid carrier, such as a fatty oil, e.g., cacao butter.

[0106] Suitable pharmaceutical excipients include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides.

[0107] In yet another embodiment, the therapeutic compound can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105).

[0108] Other controlled release systems are discussed in the review byLanger (1990, Science 249:1527-1533).

[0109] In one embodiment of the pharmaceutical composition according tothe invention, two or more active constituents are comprised as separateentities. The two entities may be administered simultaneously orsequentially.

[0110] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0111] The invention is further explained by the following illustrativeexamples.

5. EXAMPLE 1 Manufacture of Processed DIM or LTr-1 for Enhanced OralBioavailability

[0112] Preparation of processed DIM and LTr-1 was accomplished accordingto the steps outlined in U.S. Pat. No. 6,086,915. Briefly, this includedmixture of about 10-40% by final weight of either DIM or LTr-1 withabout 10-40% by final weight of vitamin E succinate polyethylene glycol1000 (Vitamin-E-TPGS, Eastman Chemical), 2-20% by final weight,phosphatidyl choline (Phospholipon 50G, Rhone Poulenc) and 15-30% byfinal weight hexanol. This mixture was made homogeneous by mixing. Thehomogeneous mixture of indoles and other oil soluble substituents listedabove was added to a solution of modified starch in water (Capsul Starchfrom National Starch, Inc.). The starch component forms form from 30-70%of the final dry weight of the product. The well dispersed finalcombined mixture was then subjected to spray drying. The resultantproduct was a fine powder containing either DIM or LTR-1 microparticlescontained within the larger starch particles.

6. EXAMPLE 2 Manufacture of Capsules Containing I3C, DIM AND LTr-1

[0113] Pure Indole-3-carbinol (I3C) was obtained from standard suppliers(Sabinsa, Inc or Designed Nutritional Products, Inc.). Capsulescontaining 300-500 mg were manufactured by placing that amount of I3Cinto opaque gelatin capsules.

[0114] Capsules containing 150-300 mg of processed DIM, as producedaccording to the steps described in Example 1, were made by mixing theprocessed DIM with microcrystaline cellulose and placing the mixedpowder into opaque gelatin capsules. In a preferred embodiment, capsulescontaining 150-300 mg of processed DIM, as produced according to thesteps described in Example 1, were made by mixing the processed DIM withmicrocrystaline cellulose and calcium carbonate and placing the mixedpowder into opaque gelatin capsules. Alternatively, purediindolylmethane (DIM) was obtained from standard suppliers (Sabinsa,Inc or Designed Nutritional Products, Inc.). Capsules containing 300-500mg were manufactured by placing that amount of DIM into opaque gelatincapsules.

[0115] Similarly, capsules containing 150-300 mg of processed LTr-1, asproduced according to the steps described in Example 1, were made bymixing the processed LTr-1 with microcrystaline cellulose and placingthe mixed powder into opaque gelatin capsules. In a preferredembodiment, capsules containing 150-300 mg of processed LTr-1, asproduced according to the steps described in Example 1, were made bymixing the processed LTr-1 with microcrystaline cellulose and calciumcarbonate and placing the mixed powder into opaque gelatin capsules.

7. EXAMPLE 3 Manufacture of DIM or LTr-1 in a Cream for TransdermalDelivery

[0116] For the aqueous phase of the emulsion, a mixture of 70 grams ofpropylene glycol and 633 grams of water is heated to 95° C. The oilphase of the emulsion is prepared by heating a mixture of the followingto 105° C.: 30 grams cetostearyl alcohol (Alfol 16/18, Vista), 30 gramshydrogenated soy monoglyceride (Myverol 18-06, Quest), 30 g of a mixtureof polyoxyethylene stearic acid ester and mono- and di-glycerides offatty acids (Arlacel 165, ICI), 10 grams polyethylene (Epolene N-34,Eastman), and 50 g of squalene. The active ingredient phase is preparedseparately also by gently heating to about 63° C. a mixture of thefollowing to uniformity: 30 g d-α-tocopherol polyethylene glycol 1000succinate (Vitamin E TPGS, Eastman), 50 g isopropyl myristate, and 15 gof DIM or 15 g of LTr-1. The above oil phase is added to the aqueousphase using a rotor/stator type homogenizer at moderate speed. Themixture is cooled to 75° C. and 50 grams of lemon oil is added with lowspeed mixing followed by addition of the active ingredient phase.Lastly, 2 g of a 3:1 mixture of methyl paraben to propyl paraben isadded to the emulsion. This mixture is transferred to the reservoir of ahigh pressure homogenizer such as the Microfluidics™ Model 110Y. Theemulsion is passed through the homogenizer approximately five times at15,000 psi operating pressure that is sufficient to form a cream of thedesired consistency which will not separate on standing.

[0117] For the aqueous phase of the emulsion, a mixture of 70 grams ofpropylene glycol and 633 grams of water is heated to 95° C. The oilphase of the emulsion is prepared by heating a mixture of the followingto 105° C.: 30 grams cetostearyl alcohol (Alfol 16/18, Vista), 30 gramshydrogenated soy monoglyceride (Myverol 18-06, Quest), 30 g of a mixtureof polyoxyethylene stearic acid ester and mono- and di-glycerides offatty acids (Arlacel 165, ICI), 10 grams polyethylene (Epolene N-34,Eastman), and 50 g. of squalene. The active ingredient phase is preparedseparately also by gently heating to about 63° C. a mixture of thefollowing to uniformity: 30 g d-α-tocopherol polyethylene glycol 1000succinate (Vitamin E TPGS, Eastman), 50 g isopropyl myristate, and 15 gof DIM or 15 g of LTr-1. The above oil phase is added to the aqueousphase using a rotor/stator type homogenizer at moderate speed. Themixture is cooled to 75° C. and 50 grams of lemon oil is added with lowspeed mixing followed by addition of the active ingredient phase.Lastly, 2 g of a 3:1 mixture of methyl paraben to propyl paraben isadded to the emulsion. This mixture is transferred to the reservoir of ahigh pressure homogenizer such as the Microfluidics™ Model 110Y. Theemulsion is passed through the homogenizer approximately five times at15,000 psi operating pressure that is sufficient to form a cream of thedesired consistency which will not separate on standing.

8. EXAMPLE 4 Manufacture of DIM or LTr-1 in a Liposomal Spray forSublingual and Mucosal Delivery

[0118] A standard liposomal preparation technique was used to prepare aliposomal suspension of DIM and separately, a liposomal preparation ofLTr-1. Briefly, propylene glycol (7.0 gms) was heated to 92° C. on awater bath, 8 grams of partially hydrogenated pure egg yolk lecithin,and 320 mg of stearylamine were added and dissolved to give a clearliquid. To this liquid was added 500 mg of jet milled DIM. Thistranslucent solution was added to 200 ml of a 1% aqueous solution ofdextran T 40 pre-warmed to 55° C. and the mixture was stirred in apropeller mixer at 50° C. for 3 minutes after which it was cooled toroom temperature. This procedure yielded an off-white, dextran T40/liposome suspension, thus encapsulating the DIM.

[0119] Equivalent steps were undertaken to prepare a lipsomal suspensionencapsulating LTr-1.

9. EXAMPLE 5 Use of Processed DIM for Treatment of Mastalgia

[0120] The purpose of this study was to determine whether dietarysupplementation with the cruicferous phytochemical, Diindolylmethane(DIM) is effective in relieving the pain of this chronic condition.

[0121] B. R. is a 44 year old woman referred for evaluation ofperimenopausal symptoms of chronic recurring breast pain. She had beentroubled by breast pain for many years with recent worsening in hersymptoms.

[0122] At age 35 she had undergone a vaginal hysterectomy due toexcessive and painful menstrual periods. Since her ovaries were leftintact, she has received no Hormonal Replacement Therapy (HRT). Duringthe last 4 years she experienced a monthly cycle of breast pain forabout one week of each month. The breast pain was bilateral, associatedwith significant tenderness to touch, and a discomfort described as“heaviness or swelling.” A trial of a women's health supplementcontaining Don Quai provided no relief.

[0123] Evaluation included a baseline morning urine sample and closeassessment of her symptoms of breast pain during the ensuing month.Following this, bioavailable DIM was begun as a supplement at 225 mg perday together with increased dietary fiber. Complete resolution of thebreast pain was noted following one month. A second urine sample wasobtained after one month on the DIM supplement.

[0124] The urine samples were subsequently analyzed for their levels of2-hydroxy and 16-hydroxy estrogen metabolites, using an Elisa basedassay (Estratest, Immunacare, Inc., Bethlehem, Pa.). These results ofbefore and after DIM supplementation breast pain scores and estrogenmetabolite testing are summarized below. Indicator Monitored Before DIMAfter DIM Typical Breast Pain Score Moderate Absent KEY: 0 = none 2/40/4 1 = mild; 2 = moderate; 3 = significant; 4 = severe Typical BreastSoreness Score Significant Absent 3/4 0/4 Urinary Estrogen Metabolites(ng/ml/mg Creatinine): 2-Hydroxy estrone level 22.2 26.4 16-Hydroxyestrone level 7.3 5.1 2-Hydroxy to 16-Hyroxy estrone ratio 2.97 5.08Total urinary estrone metabolites 28.9 31.3

10. EXAMPLE 6 Open Label Study of Oral Processed DIM in Women WithBreast Pain (Mastalgia)

[0125] 20 women were referred by collaborating physicians forparticipation in an open label study of the use of a processed DIMdietary supplementation for recurrent mastalgia. Individuals wereselected who met the criteria of recurrent bilateral breast pain for atleast 6 months. During the study, the participants avoided herbs andother dietary supplements which might effect estrogen. This includedavoidance of Evening Primrose Oil, borage oil, soy isoflavones, RedClover extract, Don Quai, and Black Cohash. Using a breast paincalendar, participating individuals identified their level of pain,soreness, and swelling on separate analog pain scale scores for eachcategory. 18 out of 20 participants showed some improvement in theirsymptoms which was noted from 10 days to 1 month after initiatingsupplementation. Of those participants showing improvement, 12 showedsignificant improvement with levels dropping from moderate and severe tomild or absent. These improvements in symptoms were seen at a dose of150 to 300 mg/day of the supplement which corresponds to a daily dose of50-100 mg/day of DIM. No participants reported side effects. Of furthernote was one participant aged 51 who had complete resolution of pain andnoted the disappearance of a breast cyst previously documented by herphysician.

11. EXAMPLE 7 Transdermal Use of DIM for Treatment of Chronic BreastPain

[0126] E. B. is a 38 year old woman with the recurrent symptoms ofbilateral painful breasts. She describes the pain as bilateral, worsebefore menstrual periods and occurring every month beginning about 1week following cessation of her menses. The pain is described as asoreness associated with movement and a feeling of heaviness or swellingof both breasts.

[0127] After maintaining a diary documenting breast pain for onemenstrual cycle, E. B. began to apply a cosmetic formulation containingDIM. After only one week of application of 5 cc of the cream nightly,complete resolution of the pain was noted. Also noted was a clearresolution in the sense of swelling of the breasts. The cream was usedconsistently for 2 months and resolution of the pain was documented on abreast pain diary. No side effects were experienced.

[0128] Following these two months, use of the cream was discontinued.Within one month, the breast pain recurred although to a lesser severitythan was documented before treatment.

12. EXAMPLE 8 The Use of DIM for Endometriosis

[0129] P. M. is a slender and athletic woman of 32 years who soughtalternatives in her management of severe endometriosis. Her symptoms ofrecurrent mid-cycle and menstrually associated pain have been diagnosedas due to endometriosis based on pelvic laparoscopy. This procedureconfirmed aggressive endometriosis with ectopic endometrial implantsremoved from the pelvis and associated with intestinal serosal spread. Ahistory of 2 years of intense pelvic pain at mid-cycle and duringmenstrual flow was reported prior to the laparoscopy. Her mother gives ahistory of painful menstrual periods starting in her 20's. She went onto develop ovarian cancer requiring radical surgery at 56 years. Amaternal aunt developed cervical cancer requiring surgery. Both motherand aunt had a peri-menopausal history of chronic, recurrent breast painwith fibrocystic changes. Like the patient, a younger sister has beentroubled with painful menstrual periods and pelvic pain leading tolaparoscopy and the diagnosis of endometriosis.

[0130] Following the patient's laparoscopy, one menstrual period wasstill associated with significant pain. Supplementation withbioavailable processed DIM was begun approximately 6 weeks following thelaparoscopy. Initial supplementation with bioavailable processed DIMprovided 300 mg/day for one month which was reduced to 150 mg/daythereafter. Since starting treatment, there was disappearance of pain atmidcycle and improvement of pain associated with menses. The patientcontinued DIM supplementation for about one year. During this timeregular menstrual periods became more comfortable, no longer requiringanalgesics. No side effects were reported.

[0131] Diagnosis of the endometriosis and reduction in pain severitycorrelated with serial levels of serum Ca-125 antigen. Ca-125 is a serummarker with documented usefulness in monitoring the activity ofendometriosis in a given patient (Pittaway D E and Fayez J A, 1986, “Theuse of CA-125 in the diagnosis and management of endometriosis”, FertilSteril 46:790). The following chart shows the association of thismarker, useful as a measure of changes in the activity of endometriosis.Patient Pre Post Follow-up Follow-up Follow up Status Surgery SurgeryVisit #1 Visit #2 Visit #3 Use of DIM NO NO YES YES YES Ca-125 69.6 54.126.4 23.2 34.0 Antigen Pain Level Severe Moderate Improved ImprovedImproved

13. EXAMPLE 9 Use of Transdermal and Processed DIM for the Therapy ofMastalgia With Associated Improvement in Cervical Dysplasia

[0132] V. H. is a 45 year old woman with a long history of fibrocysticbreasts, recurrent severe breast pain, and cervical dysplasia. Thebreast pain occurs on a monthly basis during the second half of themenstrual cycle and requires the use of analgesics like ibuprofen. Thebreast pain diminishes with onset of the menses. Abnormal pap smears ofthe uterine cervix were first noted in her mid thirties. The cervicaldysplasia was categorized as Class I cervical intraepithelial neoplasiaon a cervical biopsy taken approximately 1 year ago. V. H. began testingtransdermal DIM in a 1.5% strength breast cream for relief of monthlybreast pain. Dramatic resolution occurred over a period of 2 weeks.During this time, a reduction and disappearance of chronic vaginaldischarge which had been present and attributed to the cervicaldysplasia were also noted. Following two weeks of transdermal use ofDIM, V. H. began daily use of oral processed DIM at a dose of 50 mg perday of DIM. After two months of oral therapy, follow up pelvicexamination revealed a more normal appearing cervix. No side effectswere noted with the use of either DIM preparation.

What is claimed is:
 1. A method of treating cervical dysplasia in asubject having cervical dysplasia comprising administering to thesubject an amount of a dietary indole selected from the group consistingof DIM and LTr-1 effective to reduce one or more symptoms associatedwith cervical dysplasia.
 2. The method of claim 1 wherein the dietaryindole is DIM.
 3. The method of claim 2, wherein the DIM is suspended asmicroparticles in a starch carrier matrix.
 4. The method of claim 1,wherein the dietary indole is formulated as a cream or suppository. 5.The method of claim 4, wherein the dietary indole is administered bydirect application to the vaginal or cervical mucosa of the subject. 6.The method of claim 4, wherein the dietary indole is formulated as acream, and is administered transdermally.
 7. The method of claim 1,wherein the dietary indole is administered orally.
 8. A method ofpreventing cervical dysplasia in a subject in danger of developingcervical dysplasia comprising administering to the subject an amount ofa dietary indole selected from the group consisting of DIM and LTr-1effective to prevent one or more symptoms associated with cervicaldysplasia.
 9. The method of claim 8 wherein the dietary indole is DIM.10. The method of claim 9, wherein the DIM is suspended asmicroparticles in a starch carrier matrix.
 11. The method of claim 8,wherein the dietary indole is formulated as a cream or suppository. 12.The method of claim 11, wherein the dietary indole is administered bydirect application to the vaginal or cervical mucosa of the subject. 13.The method of claim 11, wherein the dietary indole is formulated as acream, and is administered transdermally.
 14. The method of claim 8,wherein the dietary indole is administered orally.